The hemidesmosome integrates the extracellular matrix and the keratin- containing intermediate filament network of epithelial cells in a number of tissue types. Perturbation of the hemidesmosome either by autoantibodies or in certain genetic skin diseases leads to the detachment of an epithelial sheet from the underlying connective tissue. The resulting blisters and the likelihood of infection can be devastating. The experiments outlined in this proposal are designed to provide new insights into both the assembly of and protein-protein interactions in the hemidesmosome. Molecular genetic analyses will be used to provide clues to potential protein-protein interactions. These studies will involve transfection of constructs encoding various hemidesmosomal components or portions thereof into 804G cells, a line that readily assembles hemidesmosomes in vitro, as well as into other cell types that fail to express one or more hemidesmosomal components. The fate of transfected proteins and their impact on endogenous hemidesmosomal components and the cytoskeleton will be determined by immunocytochemistry. Biochemical approaches are detailed to further characterize potential hemidesmosomal protein-protein interactions. For biochemical studies, individual hemidesmosomal elements will be purified from a bovine tissue source and their binding capabilities will be assessed in vitro in solution as well as in solid phase assays. In some assays recombinant hemidesmosome proteins will be used. In addition, cell surface receptor interactions of hemidesmosome associated laminin variants will be assessed using column chromatography. De novo assembly of hemidesmosomes in 804G cells will be monitored during re-establishment of substrate contact following suspension culture. The role of hemidesmosome associated laminins and their integrin receptors in nucleating hemidesmosome assembly in keratinocytes and in tissue explants will be studied by light and electron microscopy and by adhesion assays. Putative changes in phosphorylation of various hemidesmosomal proteins during hemidesmosome assembly will be determined in a tissue culture model system.